IL-9 aggravates SARS-CoV-2 infection and exacerbates associated airway inflammation

SARS-CoV-2 infection is known for causing broncho-alveolar inflammation. Interleukin 9 (IL-9) induces airway inflammation and bronchial hyper responsiveness in respiratory viral illnesses and allergic inflammation, however, IL-9 has not been assigned a pathologic role in COVID-19. Here we show, in a K18-hACE2 transgenic (ACE2.Tg) mouse model, that IL-9 contributes to and exacerbates viral spread and airway inflammation caused by SARS-CoV-2 infection. ACE2.Tg mice with CD4+ T cell-specific deficiency of the transcription factor Forkhead Box Protein O1 (Foxo1) produce significantly less IL-9 upon SARS-CoV-2 infection than the wild type controls and they are resistant to the severe inflammatory disease that characterises the control mice. Exogenous IL-9 increases airway inflammation in Foxo1-deficient mice, while IL-9 blockade reduces and suppresses airway inflammation in SARS-CoV-2 infection, providing further evidence for a Foxo1-Il-9 mediated Th cell-specific pathway playing a role in COVID-19. Collectively, our study provides mechanistic insight into an important inflammatory pathway in SARS-CoV-2 infection, and thus represents proof of principle for the development of host-directed therapeutics to mitigate disease severity.

SARS-CoV-2 infection is known for causing broncho-alveolar inflammation. Interleukin 9 (IL-9) induces airway inflammation and bronchial hyper responsiveness in respiratory viral illnesses and allergic inflammation, however, IL-9 has not been assigned a pathologic role in COVID-19. Here we show, in a K18-hACE2 transgenic (ACE2.Tg) mouse model, that IL-9 contributes to and exacerbates viral spread and airway inflammation caused by SARS-CoV-2 infection. ACE2.Tg mice with CD4 + T cell-specific deficiency of the transcription factor Forkhead Box Protein O1 (Foxo1) produce significantly less IL-9 upon SARS-CoV-2 infection than the wild type controls and they are resistant to the severe inflammatory disease that characterises the control mice. Exogenous IL-9 increases airway inflammation in Foxo1-deficient mice, while IL-9 blockade reduces and suppresses airway inflammation in SARS-CoV-2 infection, providing further evidence for a Foxo1-Il-9 mediated Th cell-specific pathway playing a role in COVID-19. Collectively, our study provides mechanistic insight into an important inflammatory pathway in SARS-CoV-2 infection, and thus represents proof of principle for the development of host-directed therapeutics to mitigate disease severity.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 . COVID-19 symptoms range from mild to severe pneumonia and acute respiratory distress syndrome 1 . SARS-CoV-2 infection leads to hyperactivation of immune cells, which further induces inflammatory cascade, broncho-alveolar inflammation and immunopathology 2 . Use of dexamethasone, an anti-inflammatory drug, resulted in lower mortality and severity in patients hospitalized with COVID-19 3 , indicating that immune suppression is effective in controlling the severity and mortality. In addition, we identified that SARS-CoV-2 infection, in animal model, contributes to the extrapulmonary pathologies which include cardiovascular complications and thymic atrophy 4 .
Although the precise mechanism of disease pathogenesis and lung pathology that lead to hyper-inflammatory response is not fully understood, autopsy histopathology of pulmonary samples revealed increased accumulation of eosinophils, basophils, neutrophils and perivascular and septal mast cells in COVID-19 [5][6][7] . Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19 shows that one of the prominent cell types is Mast cells 7,8 . In line with this, mast cells-derived proteases, chymase and eosinophil-associated mediators are found to be elevated in sera of COVID-19 patient and lung autopsies 5,8 . Interleukin 9 (IL-9), a common γ chain family cytokine primarily produced by Th9 cells 9 , promotes mast cell growth and function in allergic inflammation 10 . Although IL-9 plays an essential role in severe airway inflammation and bronchial hyper responsiveness in asthma and Respiratory Syncytial Virus (RSV) infection 11,12 , the role of IL- 9 is not yet identified in SARS-CoV-2 infection, and its associated immunopathology.
IL-9 induction in Th9 cells requires a distinct set of transcription factors. Pu.1 is one of the key transcription factors essential for IL-9 induction and Th9 cell differentiation 9 . Other transcription factors, Interferon (IFN) regulatory factor 1 (Irf-1) 13 , Irf-4 14 , Basic leucine zipper transcription factor, ATF-like (Batf) 15 and Hypoxia-inducible factor 1-alpha(Hif-1α) 16,17 , were found to be associated with the Th9 cells differentiation and functions. We have identified that Foxo1, a forkhead family transcription factor, is essential for IL-9 induction not only in Th9 cells but other Th subsets 18 . In addition, the role of Foxo1 is well documented in T cell homoeostasis and tolerance through interplay between effector and regulatory T cell 19 . While Foxo1 regulates Th17 [20][21][22] cell functions, it is essential for the generation and functions of Foxp3 + Tregs and Th9 cells 23 .
This study aims to investigate the role of interleukin-9 (IL-9) in COVID-19 pathogenesis. We show that hACE2.Tg mice with CD4 + T cellspecific deficiency of the transcription factor Foxo1 are resistant to severe inflammation. Further, we also observe that exogenous IL-9 increases airway inflammation in Foxo1-deficient mice, while IL-9 blockade reduces and suppresses airway inflammation in SARS-CoV-2 infection. Here we unravel the role of Foxo1-IL-9 axis in SARS-CoV-2 infection, and its associated airway inflammation and immunopathology. These findings provide important mechanistic insights in understanding role of Foxo1-IL-9 axis in COVID-19 and could pave the way for the development of host-directed therapeutics to mitigate respiratory viral illness and disease severity.

IL-9 promotes the severity of SARS-CoV-2 infection and associated lung inflammation
We show that IL-9 levels were elevated in active COVID-19 patients infected with ancestral strain during the first wave of pandemic in mid-2020 ( Fig. 1a; Supplementary Table 1). First we tested the acquisition of mutations in the ancestral strain of SARS-CoV2, we used in this study by performing sequencing followed by minor variant analysis. Minor variant analysis indicated the presence of five single nucleotide variants (SNVs) in ancestral strains of SARS-CoV-2 (Supplementary Table 2). None of these SNVs were found to be in the cleavage site of virus. Therefore it is highly unlikely to affect infectivity, lethality of the virus or weaken the virus's pathogenicity. This is supported by the experimental evidences we provided subsequently in which we found that infection of hACE2.Tg mice with ancestral strain of SARS-CoV2 causes lethality and mice become moribund post infection as reported earlier 24,25 .
To determine the role of IL-9 in SARS-CoV-2 infection, anti-IL-9 neutralizing antibody was given to ACE2.Tg mice during SARS-CoV-2 infection at indicated time points (Fig. 1d, e). As compared to SARS-CoV-2-infection and Remdesivir (RDV) treatment, ACE2.Tg mice treated with anti-IL-9 antibody treatment resulted in rescuing weight loss and lung haemorrhage with the reduction of lung viral load ( Fig. 1 f, g; Supplementary Fig. 2f, g). Similar to RDV, anti-IL-9 antibody treatment decreased immune cells infiltration, mucus production, thickening of blood vessels and mild hypertrophy and mast cell accumulation in the SARS-CoV-2-infected lungs of ACE2.Tg mice ( Supplementary Fig. 2h).
Anti-IL-9 antibody treatment decreased inflammatory cytokines, IL-9, IL-4, IL-17 and IFN-γ and replenish the CD4, CD8 and γδ T cells in the BAL ( Supplementary Fig. 3a-e) and expression of Il9, Il4, Il5, Il6, and Th9 cell-associated transcription factors, Foxo1, Stat5, Stat6, Irf4, Bat-f, Gata3, and Irf1 ( Supplementary Fig. 3f, g) together with allergic markers, chemokine (C-C motif) ligand 2 (Ccl2), CXC chemokine ligand 5 (Cxcl5), Cxcl10, (Tryptophan hydroxylase 1) Tph1, (high-affinity IgE receptor) Fcer1 in the lung tissue samples of ACE2.Tg mice (Supplementary Fig. 3h). Contrary to IL-9 neutralization, intranasal administration of exogenous IL-9 enhanced severity of SARS-CoV-2 infection in ACE2.Tg mice as indicated by weight loss, lung lesions and viral load associated with an increased frequency of mast cells, and eosinophils in the Bronchoalveolar lavage (BAL) (Fig. 1h-l; Supplementary Fig. 1a; Supplementary Fig. 4a, b). In line with this, lung histology indicated an overall increased in immune cell infiltration, and mucus production upon IL-9 treatment in SARS-CoV-2-infected ACE2.Tg mice (Supplementary Fig. 4c). Interestingly, we found that IL-9 enhanced infection of Omicron variant of SARS-CoV-2 in ACE2.Tg mice, as indicated by loss of body weight, increased lung lesions and viral load (Fig. 1m, n; Supplementary Fig. 4d) with increased infiltration of immune cells and lung tissue damage ( Supplementary Fig. 4e). We performed the minor variant analysis of Omicron stock we used in this study and identified that there are 24 SNVs. However, none of these SNVs are found in the cleavage site of the Omicron (Supplementary Table 2), indicating unlikely possibility to affect infectivity and pathogenicity of the Omicron variant is known to cause a milder infection with a reduced mortality 27 .
To address the role of IL-9 in regulating the activity of the transgenic K18 promoter, which might regulate the expression of the transgenic hACE2, we performed Western Blot experiment in which we tested the expression of hACE2 at protein level in the presence or absence of IL-9 treatment. Briefly, K18hAce2 mice were administered rIL-9 (500 ng/mice) or vehicle intranasally for 24 h before euthanising the mice. Lungs from these were homogenised in protein lysis buffer for Western Blot analysis using anti-human Ace2 antibody. Our data indicated that IL-9 treatment did not increase, as compared to control treatment, hAce2 expression in K18hACE2.Tg mice (Supplementary Fig. 5g).
To further substantiate this finding, we used the intestinal epithelial cell line, Caco2, which is known to express Ace2 30,31 . Briefly, Caco2 cell line was treated with recombinant human IL-9 (10 ng/ml) for 24 h. Post IL-9 treatment, these treated cells were lysed with protein lysis buffer for further Western Blot analysis. Our results indicate that IL-9 treatment did not enhance Ace2 expression in Caco2 cells (Supplementary Fig. 5g), suggesting that IL-9 is not able to increase expression of hAce2, and thus may not influence the expression of the transgenic hACE2 in a non-physiological manner.
Synergistic effect of anti-IL-9 antibody treatment with Remdesivir (RDV) in SARS-CoV-2 infection We further tested synergistic effect of anti-IL-9 and RDV in controlling SARS-CoV-2 infection. A suboptimal (SO) dose of anti-IL-9 antibody and RDV alone or in combination was given to ACE2.Tg mice during SARS-CoV-2 infection as indicated (Fig. 2a). As compared to the optimal dose of anti-IL-9 antibody, SARS-CoV-2-infected ACE2.Tg mice treated with SO dose of anti-IL-9 antibody and RDV effectively rescued weight loss, increased survival, reduced lung lesions and haemorrhage, and decreased viral load (lung and faecal) while SO dose of anti-IL-9 antibody or RDV alone failed to do so ( Fig. 2b-e). SO dose of anti-IL-9 and RDV, compared to SO of anti-IL-9 or RDV, treatment reduced the frequency of eosinophils, mast cells, basophils and IL-9 + , IL-17 + and IFNγ + CD4 + T cells in BAL and mediastinal and branchial lymph node of SARS-CoV-2-infected ACE2.Tg mice (Supplementary Fig. 1b; Fig. 2f-h). Lung histology indicated that combinatorial treatment of SO dose of anti-IL-9 and RDV reduced immune cells infiltration, mucus production and mast cell accumulation ( Supplementary Fig. 6a, b), indicating that anti-IL-9 antibody and RDV treatment synergistically controlled SARS-CoV-2 infection. Altogether, we demonstrated that IL-9 regulating antiviral response while promoting airway inflammation contributes to SARS-CoV-2 infection.

Foxo1-IL-9 axis is essential for SARS-CoV2 transmission
Our data indicated Foxo1 linked to SARS-CoV-2 infection and regulate viral replication and shedding, raising a possibility that Foxo1 may control viral transmission. To understand this, SARS-CoV-2-infected ACE2.Tg and Foxo1 fl/fl .CD4 Cre+ mice were cohoused at 1:1 ratio with uninfected ACE2.Tg mice for 10-12 days followed by measuring various parameters of infections (Fig. 5a). As compared to uninfected ACE2.Tg mice, uninfected ACE2.Tg mice cohoused with SARS-CoV-2infected ACE2.Tg mice develop infection while uninfected ACE2.Tg mice cohoused with SARS-CoV-2-infected Foxo1 fl/fl .CD4 Cre+ mice develop low level of infection as indicated by weight loss, lung lesions, viral load and histological score ( Fig. 5b-f). In addition, we tested whether ACE2.Tg and Foxo1 fl/fl .CD4 Cre+ mice can transmit SARS-CoV-2 infection to healthy cohoused ACE2.Tg mice. Our previous data, indicate that Foxo1 fl/fl .CD4 Cre+ , as compared to Foxo1 fl/fl .CD4 Cre-, mice are less sensitive to SARS-CoV-2 infection (Fig. 4a, b). This could be due to lesser viral replication in Foxo1 fl/fl .CD4 Cre+ mice. Keeping this in mind, we cohoused SARS-CoV2-Foxo1 fl/fl .CD4 Cre+ mice with healthy ACE2.Tg mice for a longer period (12-day post infection) to see whether additional period allows an efficient transmission of SARS-CoV-2 to healthy cohoused ACE2.Tg mice as it does in case of infected ACE2.Tg mice cohoused with healthy ACE2.Tg mice. Even though, longer period of cohousing of SARS-CoV-2 infected Foxo1 fl/fl .CD4 Cre+ mice were unable to transmit SARS-CoV-2 infection, indicating the inability Foxo1 fl/fl . CD4 Cre+ to transmit the infection ( Supplementary Fig. 12). As compared Foxo1 fl/fl .CD4 Cre+ cohoused with ACE2.Tg, ACE2.Tg cohoused ACE2.Tg mice, showed higher frequency of eosinophils and mast cells in their BAL (Fig. 5g, h). These Data together indicated Foxo1 limits viral transmission.
Foxo1 fl/fl .CD4 Cre+ mice were resistant to SARS-CoV-2 infection and associated mortality. Foxo1-medaited IL-9 causes severity of SARS-CoV-2 infection. We tested whether exogenous IL-9 makes Foxo1 fl/fl . CD4 Cre+ mice susceptible to SARS-CoV-2 infection. Intranasal administration of exogenous IL-9 makes Foxo1 fl/fl .CD4 Cre+ mice susceptible to SARS-CoV-2 infection as their overall physical activity declined (Supplementary video 2a, b). In line with this, exogenous IL-9 treatment of Foxo1 fl/fl .CD4 Cre+ , as compared to Foxo1 fl/fl .CD4 Cre+ mice without IL-9 treatment, mice show weight loss, increased lung lesions, viral load with and increased frequency of eosinophils, mast cells and IL-4 + -, IL-9 + -and IL-17 + -producing CD4 + T cells (Fig. 5i-n; Supplementary  Fig. 11a). In line with this, IL-9 treatment to Foxo1 fl/fl .CD4 Cre+ mice increased histological score as determined by various indicated changes (Fig. 5o, p). These data indicated that replenishing IL-9 in Foxo1 fl/fl .CD4 Cre+ mice makes these mice susceptible to SARS-CoV-2 infection. However, endogenous (cellular) source of IL-9 in this case was remained unclear. Since it was shown that IL-9 is primarily produced by CD4 + cells and innate lymphoid cells (ILCs) 42 , we tested the cellular source of IL-9 in SARS-CoV2 infection in ACE2.Tg mice. We tested IL-9 cytokine staining in CD4 + T cell, NK cells and ILCs from uninfected and SARS-CoV-2 infected ACE2.Tg mice. Our data suggest the IL-9 is primarily produced by CD4 + T cells; we did not find IL-9 production from NK cells and ILCs ( Supplementary Fig. 13a-d).
We further tested whether CD4 + T cell-driven IL-9 is sufficient in making Foxo1 fl/fl .CD4 Cre+ mice susceptible to SARS-CoV-2 infection. To do this, we transferred the wt CD4 + T cells from ROSA mTmG mice into the Foxo1 fl/fl .CD4 Cre+ mice; we referred these mice as wt-CD4T-Foxo1 fl/fl . CD4 Cre+ mice. The advantage of using CD4 + T cells from ROSA mTmG mice, as these CD4 + T cells can be tracked in vivo based on their expression of RFP (Red fluorescence protein). Post transferring of CD4 + T cells from ROSA mTmG mice, we infected wt-CD4T-Foxo1 fl/fl . CD4 Cre+ and Foxo1 fl/fl .CD4 Cre+ mice with SARS-CoV-2 ancestral strain of SARS-CoV-2. We found that wt-CD4T-Foxo1 fl/fl .CD4 Cre+ mice become as susceptible as wt ACE2.Tg mice while Foxo1 fl/fl .CD4 Cre+ mice were found to be remain resistant to SARS-CoV-2 infection ( Supplementary  Fig. 13e). BALF analysis suggests that IL-9 is produced from wt mTmG, but not from Foxo1-deficeint, CD4 + T cells, further confirming our findings that CD4 + T cell-derived IL-9 contributes to the susceptibility of SARS-CoV-2 infection (Supplementary Fig. 13f-h). Taken together, our Data demonstrate that IL-9 promote SARS-CoV-2 infection, and make in Foxo1 fl/fl .CD4 Cre+ mice susceptible to infection. Altogether, we identified one of the key mechanisms of T cell-mediated immunopathology in Covid19 by showing that Foxo1 and IL-9 controls two distinctive pathological features that critically contribute to the progression and severity of COVID-19-namely anti-viral pathway and airway inflammation. The role of T cells was identified in mounting anti-SARS-CoV-2 response 43 . However, an Involvement of CD4 + T cells was not identified in promoting airway inflammation in promoting primary SARS-COV-2 infection.

Discussion
In this study, we identified one of the key mechanisms of T cellmediated immunopathology in Covid19. We show that Foxo1-IL-9 axis controls two distinctive pathological features that critically contribute to the progression and severity of COVID-19 -namely anti-viral pathway and airway inflammation. Although the involvement of adaptive immune cells, particularly CD4 + and CD8 + T cells, was identified in mounting protection in primary SARS-CoV-2 infection 43 , the role of CD4 + T cells and its cytokines were not known in regulation of anti-viral response and airway inflammation in SARS-COV-2 infection.  .com). b, c Change in body weight (twoway ANOVA followed by Tukey's test) and percent survival determined by Mantel-Cox test post SARS-CoV-2 infection and treatment (n = 5); *p < 0.05, **p < 0.005); bar graph represents as a mean ± SEM. d Lung haemorrhage score at 7 dpi on a scale of 0-5, 0 is a normal pink healthy lung, and 5 is a diffusely discoloured dark red lung; bar graph represents as a mean ± SEM (n = 5 mice per group) (oneway ANOVA followed by Tukey's multiple comparison test) ***p < 0.0005, ****p < 0.0001, ns = non-significant; arrow represents dark red lung lesions. e Viral load in SARS-CoV-2-infected ACE2.Tg mice lung tissue after therapeutic treatment (n = 5 mice), and freshly collected faeces qPCR assays (n = 5 mice per group). *p = 0.0021, ***p = 0.0367, ****p < 0.0001; one-way ANOVA followed by Tukey's multiple comparison test; bar graph represents as a mean ± SEM. f Representative FACS dot plots show frequency of eosinophils, mast cells, and basophils. Bar graph represents as a mean ± SEM (n = 5 mice per group) (one-way ANOVA followed by Tukey's multiple comparison test). g, h Intracellular cytokine staining of IL-9, IFN-γ and IL-17 on CD4 + T cells (n = 5); bar graph represents as a mean ± SEM; one-way ANOVA followed by Tukey's multiple comparison test. g ***p < 0.0005; ****p < 0.0001, h *p < 0.05, ns = non-significant Source data are provided as a Source data file.
We identified an association of IL-9, a cytokine primarily produced by Th cells, in enhancing the severity of SARS-CoV-2 infection. Naïve CD4 + cells differentiate into IL-9-producing Th9 cell in the presence of TGF-β and IL-4. Although the role of IL-9 and IL-9R was well documented in allergic inflammation and atopy, their functions in respiratory viral illness were not clearly understood. IL-9 was found to be present in bronchial secretion of respiratory syncytial virus (RSV) infected infants 44 . Moreover, IL-9 was found to regulate RSV-induced immunopathology and enhance RSV infection, as depletion of IL-9 promoted RSV clearances from the lungs 45 . In line with this, we show that IL-9 was found to be increased in active COVID-19 patients. Similarly, IL-9 was found to be upregulated in SARS-CoV2-infected hamster Article https://doi.org/10.1038/s41467-023-39815-5 and ACE2.Tg mice. Our data demonstrated that the depletion of IL-9 enhances viral clearance while regulates SARS-CoV-2-induced airway inflammation and lung pathology. We further characterised the role of IL-9 in SARS-CoV-2 infection and associated pathologies in ACE2.Tg mice. Our data suggested that IL-9 was found to be increased in early days of SARS-CoV-2 infection in hamster, which further indicated the involvement of IL-9 in the induction of airway inflammation. In line with this, we found that the expression of IL-9 was found to be increased in the PBMCs of active COVID-19 patients.
Role of IL-9 in inducing airway inflammation and immunopathology is well characterized, as it was shown to greatly enhance infiltration of lymphocytes, eosinophils and associated with mast cells hyperplasia and mucus production 46 . It was previously shown that anti-IL-9 neutralization markedly decreased allergic inflammation associated with reduced levels of total serum IgE, eosinophil frequency, mucus production and overall histopathological score 47,48 . In line with this, our data demonstrated that anti-IL-9 neutralization in SARS-CoV-2 infection reduced overall of histopathological score associated with the reduction in mucus production, collagen deposition and mast cell accumulation in the lungs. In line with this, exogenous treatment of IL-9 enhanced overall histopathological score in SARS-CoV-2 infection with an increased in eosinophils, mast cell accumulation and mucus production in the lungs. Taken together, our data indicated a crosstalk between IL-9 and mast cell in SARS-CoV-2 infection, which is consistent with the previous findings that IL-9 promotes mastocytosis and mast cell functions by enhancing their survival 49,50 . Although our data suggest that IL-9 enhances the severity of the SARS-CoV2 infections, mutations in the spike protein of SARS-CoV2 affect its infectivity and antigenicity. Mutation in the multi-basic cleavage site (PRRAR) at the spike protein S1/S2 interface is a major determinant of infectivity and pathogenesis of SARS-CoV2 and its variants, as it allows the efficient cleavage of S protein by the host proteases, which is a prerequisite for efficient virus entry and fusion 51,52 . The mutation of Proline (P) to Histidine (H) P681H in the cleavage site as seen in Alpha and Omicron variants has been suggested to reduce the viral infectivity as compared to the Delta virus in which the introduction of additional basic residue P681R might be responsible for the enhancement of pathogenesis 53,54 . However, except for the Proline mutation, the cleavage site is found to be highly conserved in SARS-CoV-2 variants, and mutation of the polybasic arginine residue might result in the development of low virulent strains and infection might restrict the localized spread as compared to systemic spread as shown in Influenza viruses 55,56 . In addition, mutations in other than the spike protein like D614G, N501Y, and L452R were found to be associated with viral infectivity and antigenicity. However, we did not find these mutations in the viral stocks, we used in our study.
Our data suggested that IL-9-producing Th9 cells contribute to the progression of SARS-CoV2 infection and associated airway inflammation. However, IL-9 was also found to be produced by ILCs 42 , indicating a possibility of ILCs, in addition to Th9 cells, in contributing to SARS-CoV2 infection and associated lung inflammation. We tested the role of ILCs-mediated IL-9 production in SARS-CoV-2 infection in ACE2.Tg mice. Our data indicated that neither NK cells nor ILCs contribute to IL-9 production in the lungs of SARS-CoV2 infected mice. We previously identified that Foxo1 is one of the key transcription factors that is essential for the induction of IL-9 in Th cells 18 . Hence Foxo1 deletion may lead to the attenuation of IL-9mediated SARS-CoV2 infection and associated lung inflammation. In line with this, our data demonstrated that Foxo1 deficiency in CD4 + T cells leads to blunted IL-9 production, which makes these mice less susceptible to SARS-CoV-2 infection and associated airway inflammation. Interestingly either exogenous IL-9 or transfer of Foxo1sufficient CD4 + T cells make Foxo1-deficient mice susceptible to SARS-CoV2 infection and associated inflammation further indicating the role of Foxo1 in CD4 + T cell-derived IL-9 in driving airway inflammation in this model.
Although IL-9-mediated regulation of anti-viral function was shown in RSV infection 45 , the mechanism by which IL-9 regulates antiviral functions was not identified. It was documented that anti-viral genes and ISGs were found to be essential for the clearance of SARS-CoV-2 infection. In line with this, our data suggest that IL-9 suppresses the expression of anti-viral genes and ISGs, which could be responsible for an increased severity of SARS-CoV-2 infection in the presence of IL-9. Although there is no clear mechanism is identified as to how IL-9meidates suppression of type 1 IFN response. Our data indicated that while anti-IL-9 antibody neutralization suppressed SARS-CoV-2 infection and increased anti-viral type 1 IFN response, exogenous recombinant IL-9 treatment enhanced SARS-CoV-2 infection and suppressed anti-viral type 1 IFN response. Upon binding to its receptor, IL-9 activates proximal signalling events primarily driven by JAK-STAT pathways, which includes Jak1, Jak3 and Stat1, Stat3 and Stat5 57 . Anti-viral response of type 1 IFNs also requires activation of STAT proteins. It was shown that Stat3 negatively regulates type 1 IFNs response, as Stat3 deficient cells produced enhanced type 1 IFNs, which in turn increased ISGs expression 58 . Since IL-9 activates Stat3, it might suppress type 1 IFNs production and function and susequently ISGs expression. This is could be one of the potential mechanisms by which IL-9 may suppress type 1 IFNs response. However, this needs to be experimentally evaluated and validated. Our data suggested that increased type 1 response together with anti-viral therapy might provide alternate therapy, as anti-IL-9 antibody with anti-viral drug, RDV, reduce both viral load and lung immunopathology. In fact, suboptimal doses of anti-IL-9 and RDV synergistically eliminate SARS-CoV-2 infection and reduce lung pathologies and inflammation. Foxo1 is essential for the induction of IL-9 in Th9 cell, and other Th cell subsets 18 . Foxo1 blockade reduces allergic inflammation in asthma due to reduction in IL-9 production in the lungs 18 . In line with this, our data show that Foxo1 inhibition using pharmacological inhibitor leads to suppress SARS-CoV-2 infection and associated immunopathology. Consistently, we demonstrated that . Gene expression levels in the heat map are z score-normalized values determined from log2 transformed relative gene expression. Foxo1 (Human; n = 9) (b), Hamster (n = 3 hamster per group) (c), and mice (n = 3 mice per group) at day 2 (d) is represented in the form of a bar graph (*p < 0.05, ****p < 0.0001; student's ttest); bar graph represents as a mean ± SEM. e Pictorial diagram shows the experimental design of the study (Created with BioRender.com). f Percentage change in the body weight (n = 5 mice per group) (***p < 0.002, ****p < 0.0001; twoway ANOVA followed by Tukey's test); bar graph represents as a mean ± SEM. g Percent survival of each group analysed by Mantel-Cox test (**p < 0.0047, n = 5); bar represents as a mean ± SEM. h Relative lung viral load by qPCR assay and Lung haemorrhage scored on 7 dpi (*p < 0.05, **p < 0.0047, ****p < 0.0001); bar graph represents as a mean ± SEM (n = 5 mice per group); (one-way ANOVA followed by Tukey's multiple comparison test). i-k Left panel: Intracellular staining of IL-9 was performed (n = 4) in BAL cells; middle panel (j) shows relative mRNA expression of Il9 and right panel (k) shows the IL-9 concentration in BAL fluid by ELISA (n = 5), *P < 0.05, ***P < 0.0004, p < 0.0001 (right: Kruskal-Wallis test, left and middle: twoway ANOVA). l-n FACS plots and their respective bar graphs represent Eosinophils, Mast cells, and Basophils percentages in BALF. *p = 0.0471, **p < 0.005, ***p = 0.0002, ****p < 0.0001 (one-way ANOVA followed by Tukeys multiple comparison test) n = 5. o, p relative mRNA expression of Irf4, Batf, Smad3, Irf3,Irf7, and Irf9 (*p = 0.0406, **p = 0.0060, ***p = 0.0003, ****p < 0.0001; one-way ANOVA). q, r relative mRNA expression of ISGs (Ifitm, Ifnβ, Ifnar1, Ifnar2, Ifnα) and anti-viral factors genes (Oas2, Oas3, Oas1g, RNaseL, and Adar) profile by qPCR from lung samples (n = 4); *p < 0.05, **p < 0.005, ***p < 0.005, ****p < 0.0001 (one-way ANOVA followed by Tukey's, multiple comparison test); bar graph represents as a mean ± SEM. Source data are provided as a Source data file.
Foxo1-conditional deficiency in CD4 + T cells make ACE2.Tg mice resistant to SARS-CoV-2 infection associated with the blunted IL-9 production. Mechanistically, similar to IL-9 neutralization, Foxo1 deficiency in CD4 + T cells leads to the upregulation of anti-viral and ISGs. This could be due to the Foxo1-dependent regulation of anti-viral function by promoting Irf3 degradation 59 . Importantly, both IL-9 neutralization and Foxo1 conditional deficiency in ACE2.Tg mice generate a similar phenotype in terms of immunopathology in SARS-CoV-2 infection associated with the accumulation of eosinophils, mast cells in the lungs and other related pathologies. Taken together, our data demonstrated that Foxo1-IL-9 axis regulates SARS-CoV-2 infection and associated immunopathology.

Human ethics
The study was approved by the Institutional Ethics Committee (Human Research) of THSTI and ESIC Hospital, Faridabad (Letter Ref No: THS 1.8.1/(97) dated 07 July 2020). After obtaining an approval from the Institutional Ethics Committee of THSTI (IEC, Human Research) and ESIC Hospital, Faridabad active COVID-19 patients' blood samples were collected from symptomatic COVID-19 patients and healthy participants after the written informed consent and there was no bias to the recruitment or collection. PBMCs were isolated from collected blood samples, and stored in liquid nitrogen as previously described for further use 18 . Briefly, human PBMCs were isolated by Ficoll Gradient (GE Healthcare).
In vivo treatments of RDV, anti-IL-9, Foxoi and exogenous IL-9 All the studies were designed to study therapeutic efficacy as compared to RDV. Therapeutic studies (Neutralisation experiments) were performed to define if drug regimens could affect virus replication and disease progression. 6-8-week-old mice (Male and Female) were used in this experiment. All treatments were initiated one day prior to infection. Post challenge, uninfected and infected mice were observed till the infected mice lost 20-30% weight and the animals were sacrificed when they became moribund for further validation. In two other experiments, treatment with a vehicle, RDV (25 mg/kg; ip), anti-IL-9 (20 μg/mice; ip), and Foxo1 inhibitor (20 mg/Kg; i.n.) were given. On appearance of moribund features, animals were euthanized by isoflurane; lungs were scored for haemorrhage (described below). The left lobe was placed in 10% formalin and stored at room temperature until sectioning and histological analysis. Lung sectioning, haematoxylin, eosin staining, and N antigen staining as described below was performed at the Institute of Liver and Biliary Sciences (ILBS) at New Delhi.
To evaluate the role of IL-9, we used Foxo1 fl/fl .CD4 Cre+ mice compared to hACE2.Tg mice infection with rIL-9 i.n. (i.n.; 500 ng/mice; 6-8week-old mice (male and female) were used in this experiment.). Euthanasia and immunopathological assays were performed as described above.
Cohousing and viral transmission hACE2.Tg mice (6-8 weeks male and female) and Foxo1 fl/fl . CD4 Cre+ ACE2.Tg (6-8 weeks male and female) mice were infected with the with SARS-CoV-2. 24 h post infection, infected mice were cohoused with the uninfected ACE2.Tg mice (1:1 ratio). Subsequently, mice were followed for the sign of infection and other parameters were measured.

Measurement of viral load
Lung tissues and faecal samples were weighed and homogenized for further processing as described earlier 60 . Briefly, RNA was extracted by using Total RNA Isolation Kit (MDI) as per the manufacturer's protocol. Relative copy number of SARS-CoV-2 RNA was done using previously used formula (POWER (2, −ΔCT)*10,000 to calculate the relative gene expression 60 . cDNA was synthesized using the kit from applied biosystem. Copies of SARS-CoV-2 nucleocapsid (N) RNA were determined using the N gene primers (forward: 5′-ATGCTGCAATCGTGCTACAA-3′; reverse: 3′-GACTGCCGCCTCTGCTC-5′. β-actin gene was used as an endogenous control for normalization. ΔΔCt method was used for relative quantitation 18,61 .

Peripheral blood mononuclear cells (PBMCs) isolation
PBMCs were isolated using density-gradient centrifugation as described previously 18 . Briefly, human PBMCs from healthy donors were isolated by Ficoll-paque (GE Healthcare) gradient, and the PBMCs were then washed once with 1X PBS, followed by isolation of total RNA using .Tg (RDV group as a control) was intranasally challenged with SARS-CoV-2 and euthanization at 7 dpi. a Percentage changes in body Weight (n = 5; two-way ANOVA followed by Tukey's multiple comparison test; *p = 0.0366, ***p = 0.0001; bar graph represents as a mean ± SEM. b Percent survival by Mantel-Cox test (***p = 0.0095). c Image shows gross lung morphological changes d Relative viral load was measured by qPCR (****p < 0.0001); bar graph represents as a mean ± SEM (n = 4 mice per group); one-way ANOVA followed by Tukey's multiple comparison test. e, f BALF cells were stimulated with PMA/Ionomycin and IL-9, IL-4 production was determined by flow cytometry (*p < 0.05, **p < 0.005, ****p < 0.0001; n = 5) (one-way ANOVA followed by Tukey's multiple comparison test); bar graph represents as a mean ± SEM. g-j Heat maps of significantly up and downregulated genes during SARS-CoV-2 infection. g Heat map of all significantly differentially expressed top 100 genes DEGs (differentially expressed genes) and further heat-map analysis of selected top significantly differentially expressed genes. h Fibroblast genes i antiviral & asthma, lung injury genes in SARS-CoV-2, j allergy, Type I IFN signalling genes identified through DEG analysis. Genes shown in each pathway are the union of the differentially expressed genes from Wt (hACE2.Tg) Vs. Foxo fl/fl .CD4 Cre+ comparisons. Columns represent samples, and rows represent genes. Gene expression levels in the heat maps are z score-normalized values determined from log2 values. Source data are provided as a Source data file and RNA seq data available at NCBI.
Trizol reagent. The RNA isolated from PBMCs was used to test the expression respective genes relative to β-Actin.

Gene expression profiling of human PBMCs
Peripheral blood mononuclear cells (PBMCs) were isolated using density-gradient centrifugation as described previously 62 . Briefly, blood samples were collected from symptomatic COVID-19 patients (~0-3 days from PCR positive report) and healthy volunteers in heparinized CPTTM (BD Biosciences, USA) tubes and were centrifuged at 1500 × g for 25 min. The PBMC layer was separated and washed with 1X PBS, and total RNA from PBMCs was isolated using the Trizol reagent. The RNA isolated from PBMCs was used to test the expression Article https://doi.org/10.1038/s41467-023-39815-5 of respective genes relative to β-Actin by RT-PCR. The relative expression levels (2 −ΔΔCt ) of genes were further normalized by log2 transformation, and Z-score was calculated as described previously 63 . The median of Z-scores of log2 transformed relative gene expression was represented as a heat map.
qPCR RNA was isolated from lungs and spleen as described previously 18 . RNA from BAL cell samples was isolated by using RNAeasy kit (MDI). Total RNA was subjected to cDNA synthesis using the iScript cDNA synthesis kit (Biorad; #1708891). qPCR was performed as described earlier using SYBR green dye KAPA SYBR FAST qPCR Master Mix (2X) Universal kit on standard 7500 Dx real-time PCR system (Applied Biosystems). The relative gene expression was calculated as described previously 18

BALF collection and lung histological analysis
Lung lavage was collected by inserting a cannula into the trachea and lavaging with 500 μl cold PBS three times as previously described 12 .
Lavage sample was centrifuged, and supernatant was collected for further cytokine analysis, and cells were used for FACS analysis. Lungs were excised; left lower lobe was immersed in 10% formalin and used for histological analysis. Paraffin-embedded tissue samples were further sectioned and various histological staining's (H&E, Periodic acid Schiff's, van Gieson, and toluidine blue) were performed at ILBS (New Delhi) and the scoring was done by a trained histopathologist independently in a blinded manner.

Immunohistochemistry
For immunohistochemistry 2-4-μm sections were used as described earlier 12 . Briefly, paraffin-embedded sections were dewaxed and rehydrated through xylene and graded alcohol, respectively, for 15 min at room temperature (RT), before epitope unmasking, slides were blocked with normal goat serum for 30 min in RT. Samples were then incubated with a primary antibody incubated overnight at 4°C (SARS-CoV-2-N antigen (5-25 μg/ml; R&D, # Clone # 1035145)). Speciesmatched gamma globulin was used as an isotype control at the same concentration. Sections were washed in PBST and Species-matched secondary antibodies were applied for 60 min at RT. The finally stained sections for SARS-CoV-2-N protein were then observed and images were captured under Ti Eclipse Nikon microscope at RCB (Regional Centre for Biotechnology).

Cytokine ELISA
Quantitation of IL-4, IL-9, IL-10 and IFN-γ was measured in BALF ELISA as described earlier 12 . Briefly, ELISA plates were coated overnight by anti-IL-4, anti-IL-9, anti-IL10 or anti-IFNy antibodies overnight in bicarbonate buffer. Thereafter, wells were washed and blocked and then incubated with BAL fluid at 1:1 dilution. The wells were then washed and incubated with detection antibody conjugated with biotin. Colour was developed by incubating with Avidin-HRP enzyme and then with TMB substrate. Reaction was stopped by using 0.2 N stop solution and plate was read at 600 nm in spectrophotometer (BioLinkk). For sorting of mTmG CD4 + , Foxo fl/fl .CD4 Cre+ CD4 + T cells, we sacrificed the ROSA mTmG wt mice and Foxo fl/fl .CD4 Cre+ mice euthanized; spleen and lymph nodes were collected aseptically and singlecell suspensions were made from 6-8-week-old mice. CD4 + T cells were purified using anti-mouse CD4-Percp cy5.5 (#100538; Biolegend). CD4 + T cells were further sorted using fluorescence-activated cell sorting (FACS) on BD FACSAriaIII (BD Biosciences) to obtain PE + CD4 + T cells and CD4 + T cells using anti-CD4-Percp Cy5.5. The purity of sorted cells was typically~96% in post-sort analysis.

hACE2 expressing Intestinal epithelial cells isolation (IECs)
Primary IECs were isolated as described earlier 64 . Briefly, the intestine from the ACE2.Tg mice was cleaned with the cold PBS containing Gentamycin The intestine was cut longitudinally and mucus layer was removed and placed in EDTA (30 mM) solution at 4°C for 20 min. Colon epithelial cells were gently removed and washed with PBS. Subsequently, the collected cells were plated in collagen-coated plates.

Western blot
We treated the hACE2.Tg mice with or without rIL-9 (500 ng/mice; i.n.). After 24 h, we sacrificed the mice using an overdose of Ketamine and Xylazine. Cardiac perfusion was performed, and further, we digested the lungs with Dispase to get the single-cell suspension. We collected the cells and lysed them in RIPA buffer containing PIC (Protease inhibitor cocktail). In line with this, we treated the Caco2 cells (Human epithelial cell line) with or without rIL-9 (10 ng/ml) for 24 h. We have lysed the cells in RIPA buffer, as mentioned above. The protein concentration was determined by performing a BCA protein assay (Bio-Rad). 40 µg of protein extracts were loaded on a 10% SDS-Gel for hACE2. Proteins were transferred to a membrane, blocked with 5% BSA, and then incubated in primary antibody (MA5-31395; Thermo-Scientific) overnight at 4°C. The HRP-conjugated anti-mouse (#7076; CST) secondary antibody was incubated for 1 h. bands were captured on Gel-doc (BIO-RAD). Band intensities were normalised with β-Actin (#4967; CST) and calculated by using ImageJ software.
Transcriptome profiling using RNA quantification sequencing RNA sequencing of SARS-CoV-2 infected lungs of ACE2.Tg, hACE2.Tgx Foxo [ .CD4 Cre-, and Foxo fl/fl .CD4 Cre+ SARS-CoV-2 infected lung tissues were homogenised and RNA was derived and subjected to nextgeneration sequencing (NGS) to generate deep coverage RNASeq data. Size selection of RNA fragments was done with SPRI Beads-based Size Selection. High-quality libraries were prepared using NEB Next Ultra II Directional RNA Library Prep Kit according to manufacturer's protocols and paired-end reads of 151 bp read length were generated on the Illumina Novoseq 6000 platform.

Transcriptome analysis
Quality-based filtering and adaptor trimming of the raw sequencing reads was done using fastp (v0.20.1). A thresh hold of 30 was set for the phred quality score. The filtered reads were aligned against the Mus musculus (mm39) genome using the splice aware aligner Hisat (v2.2.1).
The alignments were assembled into transcripts with stringtie assembler (v2.1.5). Stringtie computes read counts for the genes and normalized expression values with the Transcript per million (TPM) metric. The gene read counts were used for differential analysis between the conditions. Genes having a p-value of less than 0.05 were considered to have a significant differential change in the expression between the conditions. A log2Foldchange of 2 and higher of these significant genes were classified as upregulated and a log2Foldchange of −2 and lower as downregulated. Genes were functionally annotated with Gene Ontology terms and Reactome pathways using NCBI resources. David Bioinformatics resources (v6.8) was used to identify significant enrichment of significant GO terms and pathways. String database was used to determine for interaction of the protein-coding genes. A high confidence score of 0.9 was used to compute the interactions. R packages used for visualization -Complex Heat map, Enhanced Volcano, ggplot2.

Principal component analysis
High-dimensional expression data are mathematically reduced to principle components that can be used to describe variation across samples in fewer dimensions to allow human interpretation. Principle component 1 (PC1) accounts for the most amount of variation across samples, PC2 the second most, and so on. These PC1 vs PC2 plots are coloured by sample annotation to demonstrate how samples cluster together (or not) in reduced dimensional space. PCA emphasizes variation and brings out strong patterns in a dataset. It's used to make data simpler to explore and visualize.

Viral RNA isolation and qRT-PCR
Viral RNA from stock solutions was isolated using Trizol, and SARS-CoV-2 detection and quantification were performed using a SARS-CoV-2 kit (Illumina, Cat. No. 20044311) with a cycle threshold of 35.

Library preparation, sequencing
Whole genome sequencing of the SARS-CoV-2 and B.1.1.529 samples, using the capture-based Illumina Respiratory Virus Oligo Panel (RVOP), was done to capture the SARS-CoV-2 genome. The library preparation protocols for RVOP have been previously described. Briefly, doublestranded cDNA was prepared from 300 ng RNA using the COVID Seq kit (Cat. No. 20051772). The RVOP library was prepared using Illumina DNA Prep (Illumina, Cat. No. 20044311). Agilent 2100 bioanalyzer was used to check the quality of both libraries. The RVOP library was denatured and diluted to optimal loading concentration for sequencing on the MiSeq platform using a v3 reagent kit at 2 × 75 bp read length. The sequencing data analysis was performed as previously described. The sequencing data analysis was performed as previously described [65][66][67] .

Minor variant analysis
The primer-free pair raw reads of SARSCoV2 were generated from the Illumina MiSeq. Raw reads of SARSCoV2 were pre-processed based on read quality and read length (phred quality ≥30 and minimum length ≥50 base pair) and merged by PEAR programme 68 . The merged reads were mapped to Wuhan's SARSCoV2 sequence (Genebank ID: NC_045512.2) to generate a consensus genome 69 . During the mapping of reads to reference genome sequence, a BAM file was generated by Samtools 70 . This BAM file was processed by diversiutils script in DiversiTools (http://josephhughes.github.io/btctools/) to find the frequency of all types of four bases for each position of a reference sequence. The only variants that have been covered by at least 15 times (read depth ≥ 15) by high-quality reads (average read's phred score ≥ 30) to find highly accurate single nucleotide variants (SNVs) 71 .

Statistical analysis
All the results were analysed and plotted using GraphPad Prism 8.0 software. Percentage change weight, relative gene expression, lung haemorrhagic scores, FACS, ELISA, and qPCR studies were compared and plotted as mean using graph pad. Dataset was analysed by using one-way ANOVA, two-way ANOVA, Wilcoxon test or Student's t-test. Differences were considered statistically significant with a p value of less than or equal to 0.05.

Statistics and reproducibility
No statistical method was used to predetermine sample size. No data were excluded from the analyses. Mice of different genotypes were randomly assigned to treatment groups throughout the study. For experiments involving genetically modified animals, littermates were used for each experiment. In the cell and animal experiments, investigators were not blinded to group allocation because the investigators should give the drug to the mice and cell in different treatment conditions. Bio render software was used for pictorial representations.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
The RNA sequence Data generated in this study has been deposited in the NCBI SRA database under the accession code no. PRJNA842504. Publicly available data with accession code, GSE209550. The authors declare that, the necessary data required to validate the findings of the paper can be found within the article itself or in the Supplementary Materials. Source data are provided with this paper.